Methods and compositions for inhibiting mycotoxin contamination in cereals

ABSTRACT

We search for a cultivation method for reducing the amount of mycotoxin contamination in wheat which has been an important pending question for the quality in actual producing field of wheat and the health hazard risk for customers. 
     The present invention discloses a method of reducing the contamination amount of mycotoxin in cereals characterized in that one or more compounds A selected from the group consisting of ammonium salts, primary to quaternary ammonium salts, alkali metal salts, alkaline earth metal salts and polyvalent metal salts of phosphorous acid and phosphite ester are given to the cereals.

TECHNICAL FIELD

The present invention relates to a treatment method which intends toreduce contamination amount of mycotoxin produced by plant pathogenicfungi of cereals (hereinafter represented as “DON”) in the cereals byusing a composition for agri-horticulture containing one or morecompounds A selected from the group consisting of ammonium salts,primary to quaternary ammonium salts, alkali metal salts, alkaline earthmetal salts and polyvalent metal salts of phosphorous acid and phosphiteester as an active ingredient(s).

BACKGROUND ART

Fusarium head blight in cereals frequently occurs in cases of continuouscloudy weather and light rain, and furthermore high temperature from theheading date to the milk-ripe stage (Yukio Ozeki, Hiroshi Sasaki, YoichiAmano, Hokkaido no Hatasaku Gijutu (Farming Technology in Hokkaido)—Cereals version—, published by Society for Agricultural TechniquePropagation, page 209, 1978), and it is a plant disease which causesgreat damage to cereals in terms of yields, quality and so forth, andcan not be avoided in this country due to the high amount of rainfallduring the ripening period. As major pathogenic fungi thereof, Fusariumgraminearum, Fusarium culmorum, F. avenaceum and Microdochium nivalehave been specified. Although there is difference in occupancy dependingon climate conditions and areas, complex infection is often observed infarm fields where the disease has occurred (Kunihei Kishi, NipponShokubutsu Byogai Daijiten (Comprehensive Dictionary of Plant Disease inJapan), published by Zenkoku Noson Kyoiku Kyokai (Association forNational Farming Village Education), page 74, 1998).

The plant pathogenic fungi which cause this disease produce more thanone toxic metabolites referred to as mycotoxin, which contaminates cropsduring cultivating and poses a risk of ingestion by human and domesticanimals through migration to harvestries and processed foods. Researcheson mycotoxin in areas such as Europe, North America and East Asia wherecultivation of the cereals is active have long history, DON has beenspecified as a toxic agent whose effects on human and animals are mostconcerned in terms of both toxicity and the contamination amount, andnoticed worldwide. Ingestion of foods contaminated with DON causes acutepoisoning where digestive organ symptoms including emesis and diarrheaare major symptoms. In Europe and North America, self-regulating valuesof DON contamination amount in grains have been established, and thesystem for intensified surveillance has been organized. Whileinternationally rising momentum in DON surveillance, in 2002 in thiscountry, the Ministry of Health, Labor and Welfare also established aninterim standard value of 1.1 ppm for the DON contamination amount inthe wheat, and announced that safety of the wheat distributed in themarket was assured (Shokuhatu No. 0521001). In relation to theannouncement, as an instruction notice for feed safety, the Ministry ofAgriculture, Forestry and Fisheries established the interim acceptablevalue 4.0 ppm for deoxynivalenol in feeds supplied for cattle aged 3months or more and 1.0 ppm that for domestic animals other than theabove (press release). Before DON has been noticed as a fungus producingtoxin, people have been protected from healthy risk due to fungous toxinby a law regulation that the contamination ratio of the mixed grainsaffected with Fusarium head blight shall be made less than 1% by visualcheck at the shipping stage of crude wheat. Thus, in the productionfields of the cereals, the damages with Fusarium head blight have beenreduced and inhibited by giving fungicidal agents effective forpathogenic fungi of Fusarium head blight.

Chemicals effective for the pathogenic fungi of Fusarium head blight ofthe cereals commonly used at present are classified into several groupsby their action mechanisms and chemical structures of active ingredients(Agricultural Chemical Handbook, 2001 version, published by NipponShokubutsu Boeki Kyokai). The SBI agents characterized by inhibition ofbiosynthesis of sterol universally present as a component of biologicalmembrane of fungi include(RS)-1-p-chlorophenyl-4,4-dimethyl-3-(1H-1,2,4-triazol-1-ylmethyl)pentan-3-ol(common name: tebuconazole),(1RS,5RS)-5-(4-chlorobenzyl)-2,2-dimethyl-1-(1-H-1,2,4-triazol-1-ylmethyl)cyclopentanol(common name: metconazole), and1-[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-ylmethyl)-1H-1,2,4-triazole(common name: propiconazole), and they are characterized by having atriazole skeleton in the chemical structures. They have been widely usedsince they are effective at low doses, rapidly permeate in plant bodies,are resistant to rain and low toxic for flower-visiting insects, andthey have a high protection effect on the pathogenic fungi of Fusariumhead blight of the cereals. Methyl=(E)-2-(2-(6-(2-cyanophenoxy)pyridine-4-yloxy]phenyl)-3-methoxyacrylate (common name: azoxystrobin)and methyl=(E)-2-methoxyimino [α-(o-tolyloxy)-o-tolyl]acetate (commonname: kresoxim-methyl) which were developed as strobilurine derivativeswhich are anti-mycotic antibiotics and structurally characterised bymethoxyacrylate ester are classified into methoxyacrylate typefungicidal agents. The former exerts the protection effect by theinhibition of respiratory activity of the fungi, and the latter does itby the inhibition of cytochrome electron transfer system inmitochondria. The other synthetic fungicidal agents include1,1′-iminodi(ootamethylene)diguanidium-triacetate (common name:iminoctadine acetate). It is considered as the action mechanism that itcauses destruction of a membrane lipid bilayer structure of fungi due toa surfactant-like feature derived from its salt structure. Furthermore,the relevance between the pathogenic fungi and drug efficacy has beenresearched, and it has been demonstrated that the triazole agents areeffective for the 3 species of F. graminearum, F. culmorum and F.avenaceum, and that the methoxyacrylate agents are effective for M.nivale. As mentioned above, occurrence of the disease often accompaniesmixture of the multiple pathogenic fungi, and thus, the occurrence ofthe disease has been protected and inhibited by performing rotationspray with taking advantage of characters of respective agents.

Along with attention for DON, as analysis of its contaminationconcentration has progressed, it has been being demonstrated that thereis no direct relevance between an illness degree of the Fusarium headblight and the contamination amount of DON (Rai, G. H., Plattner R andDesjardins A., Relationship between visual scab rating anddeoxynivalenol in wheat cultivars, The 1988 National Fusarium HeadBlight Forum, Chapter 2, pages 21-25). It has been also demonstrated inrecent years that F. graminearum and F. culmorum have DON productioncapacity but F. avenaceum and M. nivale do not produce DON. It has beenreported that when M. nivale is controlled using the methoxyacrylateagent, F. graminearum and F. culmorum which are antagonized areincreased resulting in facilitating the increase of DON contaminationamount. Thus, the disease protection only by giving the fungicidalagents can not sufficiently prevent the DON contamination. Furthermore,in the production fields of the cereals, even when the Fusarium headblight of the cereals is controlled by combined application of variousfungicidal agents, DON is frequently detected at more than 1.1 ppm. Thistroubles the producers. That is, the DON contamination amount can not besufficiently reduced only by controlling the pathogenic fungi having amycotoxin production capacity, which is a conventional technology. Alsowith respect to fosetyl described in the present statement, thefungicidal effect on the plant pathogenic fungi has been known publicly(U.S. Pat. No. 4,139,616, 1979; and JP 62-87504 A), but there is nomention at all for effects on plants contaminated with mycotoxin. Undersuch a circumstance, it is an actual state that establishment of amethod of substantially reducing the DON contamination amount has beenstrongly desired in the production fields of the cereals.

The present inventors studied on compositions for agriculture whichreduces contamination of wheat with mycotoxin, particularly a DONcontamination amount to 1.1 ppm or less or a low contamination amount aspossible. As a result, a composition for agri-horticulture developedusing one or more compounds A selected from the group consisting ofammonium salts, primary to quaternary ammonium salts, alkali metalsalts, alkaline earth metal salts and polyvalent metal salts ofphosphorous acid and phosphite ester as an active ingredient(s) has alow controlling effect on pathogenic fungi of Fusarium head blight ofthe cereals but has an excellent contamination inhibitory effect on amycotoxin contamination, particularly the DON contamination, and havecompleted the invention.

Also, the inventors have shown a reduction of the DON contaminationamount equal to or less than the standard value in mixed use with afungicidal agent where DON contamination at high concentration of morethan the standard value 1.1 ppm has been observed in the mixed use withfungicidal agent for agri-horticulture, and a further inhibitory effecton the DON contamination amount in the mixed use with a fungicidal agentwhich originally shows a low contamination level, have found an effectwhere the DON contamination amount is further reduced compared to asingle treatment of a fungicidal agent for agri-horticulture, and havecompleted the invention.

DISCLOSURE OF THE INVENTION

The present invention is a method of reducing the contamination amountof mycotoxin (particularly deoxynivalenol) in cereals characterized inthat one or more compounds A selected from the group consisting ofammonium salts, primary to quaternary ammonium salts, alkali metalsalts, alkaline earth metal salts and polyvalent metal salts ofphosphorous acid and phosphite ester are given to the cereals.

Also, the invention relates to a method of reducing a contaminationamount of mycotoxin (particularly deoxynivalenol) in cerealscharacterized in that a combination of the above compound A is given tothe cereals in combination with (suitably as a composition containingboth as active ingredients) one or more fungicidal active components foragri-horticulture (hereinafter referred to as the compound B).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents test results of inhibitory effects on DONcontamination by phosphite derivatives and alkyl phosphite derivatives(Example 1).

FIG. 2 represents test results of mixed use effects of potassiumphosphite with various fungicides (Example 3).

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the invention is described in detail

The ammonium salts, primary to quaternary ammonium salts, alkali metalsalts, alkaline earth metal salts and polyvalent metal salts ofphosphorous acid and phosphite ester, derived from phosphorous acid arenot particularly limited so long as they reduce the contamination withmycotoxin, particularly DON, and for example, include alkali metal saltsand polyvalent metal salts of phosphorous acid and phosphite ester, andare suitably potassium phosphite and an aluminium salt (common name:fosetyl) of tris(ethylphosphonate). Methods of assessing the inhibitoryeffect on mycotoxin contamination, particularly DON contamination inwheat of the invention can includes a method of determining or measuringby comparing a contamination concentration of DON, incidence rate ofFusarium head blight for panicles and incidence rate for spikelets inwheat, between a group where a single treatment with phosphorous acidand phosphite ester derivative was given, and a group where a treatmentwith mixed use of other fungicidal composition was given, and a controlgroup without the treatment with drug, of the wheat cultivated in thefield.

It has been confirmed by such a method that the ammonium salts, primaryto quaternary ammonium salts, alkali metal salts, alkaline earth metalsalts and polyvalent metal salts of phosphorous acid and phosphiteester, derived from phosphorous acid have an excellent inhibitory effecton the mycotoxin contamination, particularly DON contamination,independently on controlling of the pathogenic fungi of the cereals.

The compound B in the invention could be a usual fungicidal activecompound for agri-horticulture, and is suitably the fungicidal activecompound effective for wheat Fusarium head blight, such as a sterolbiosynthesis inhibitor (SBI) having a triazole skeleton, azoxystrobin,kresoxim-methyl and iminoctadine.

EXAMPLES

By citing below test examples using potassium phosphite and an aluminiumsalt (common name: fosetyl) of tris(ethylphosphonate) as the compounds Awhich are the active ingredients of the invention, the invention is morespecifically described, but the invention is not limited thereto.

Example 1 Single Use (±Conventional Control) Effect

Wheat (cultivar: Haruyutaka) was seeded on Apr. 19, 2002, and cultivatedaccording to a conventional cultivating standard (Hokkaido, AgricultureDepartment, 1995) to establish test sections with 6:75 m² per section (3repeats). As the compounds A, potassium phosphite and an aluminium salt(common name: fosetyl) of tris(ethylphosphonate) were given, watersolution of 0.038 to 0.120% in P₂O₅ was prepared, and 100 L per 10 a wassprayed on leaves at next growth stage. That is, the first spray (June28, flowering date), the second spray (July 8, days after the floweringdate, and the third spray. (July 18, 20 days after the flowering date)were performed. A conventional control for the purpose of controllingplant pathogens and plant insects was performed together. That is, thefirst [June 20, azoxystrobin (2000 times dilution)+fenitrothion (1000times dilution)), the second [tebuconazole (Jun. 28, 2000 timesdilution)+sumithione (1000 times dilution)], and the third [July 8,propiconazole (2000 times dilution)+fenitrothion (1000 times dilution)],the forth [July 17, tebuconazole (2000 times dilution)+fenitrothion(1000 times dilution)] were performed. As controls, the test sectionwhere only the conventional control was given and the test sectionwithout any control were established. Harvest was performed byharvesting the wheat in 4 m² in the test sections on August 9 (52 daysafter the heading date). After the harvest, the wheat was applied on arice grader with 2.2 mm opening of screen to make selected grains, whichwere then pulverized by a rapid pulverizer to make whole grain powder.The whole grain powder was used as a sample for analysis. The DONcontamination concentration was analyzed by ELISA method usingcommercially available Rida Screen Fast Don supplied from r-Biopharm.

Preparation of Analysis Sample Solutions and Analysis procedure arebriefly described.

-   -   To 5 g of the whole grain powder, 100 ml of water was added, and        vigorously agitated for 10 min to make a DON extract solution.    -   The DON extract solution was centrifuged at high speed, and the        supernatant was used for the ELISA analysis.    -   According to the method described on ELISA kit, various reagents        were added, and subsequently an absorbance of each test solution        was measured.    -   The DON concentration of each test solution was read out from a        standard curve made using DON standard solutions.    -   The incidence rate of Fusarium head blight for panicles was        calculated by counting the number of panicles in 1 m² of each        section and the number of diseased panicles included therein.

The results of the present test are shown in Table 1.

TABLE 1 Inhibitory effect of phosphite derivative and alkyl phosphitederivative on DON contamination (2002, Haruyutaka) DON Incidence rate ofTreatment concen- Fusarium head Active Concen- Control tration blightfor ingredient A tration system (ppm) panicles (%) 1 Potassium 0.038%Conventional 2.41 0.3 phosphite control 2 0.070% Conventional 0.59 0.0control 3 0.112% Conventional 0.39 0.9 control 4 No control 0.69 1.2 5Fosetyl 0.120% Conventional 0.84 0.0 control 6 Potassium 0.112%Conventional 4.66 0.0 phosphate control 7 Conventional 6.16 0.9 control8 No control 8.69 4.9 Note) The concentration of the active ingredient Ais the concentration converted into P₂O₅. Note) The incidence rate ofFusarium head blight for panicles was calculated by counting the numberof panicles in 1 m² of each section and the number of diseased paniclesincluded therein.

DON was detected at highly contaminated levels which were much higherthan the interim standard value, 1.1 ppm that the Ministry of Health,Labor and Welfare had presented from the section with no control and thesection with conventional control alone using the fungicidal compositionfor controlling of Fusarium head blight of the cereals. On the otherhand, inhibition of DON contamination depending on treatedconcentrations was observed in the sections treated with potassiumphosphite and the aluminium salt (common name: fosetyl) ofTris(ethylphosphonate), and their contamination levels were lower than1.1 ppm. It is worthy of special mention that even in the sectionwithout any control where the conventional control is not performed, theDON contamination concentration is sufficiently reduced by givingpotassium phosphite. Furthermore, even when potassium phosphate which isa salt of normal phosphoric acid is given, the DON inhibitory effect islow, and thus it is obvious that the phosphite and alkyl phosphitederivatives play an important role. Even when the incidence rate ofFusarium head blight for panicles was at almost the same degree, DON athigh concentration was detected in the section without treatment withpotassium phosphite. This way, it is evident that the concentration ofDON contamination is reduced by the treatment with phosphorous acid andalkyl phosphorous acid and the derivatives thereof.

Example 2 Toxin-Inhibitory Effect by Seeds Diseased with Fusarium HeadBlight in Example 1

Healthy grains and grains diseased with Fusarium head-blight werescreened from the crude wheat obtained in Example 1, and the DONcontamination concentration of the whole grain powder thereof wasanalyzed by the ELISA method in the same way as shown in Example 1.

The results of the present test are shown in Table 2.

TABLE 2 DON contamination concentration of grains diseased with Fusariumhead blight DON concentration Treatment content (ppm) Active Concen-Control healthy diseased ingredient A tration system grain grain 1Potassium 0.038% Conventional 0.05 76.6 phosphite control 2 0.070%Conventional 0.04 44.7 control 3 0.112% Conventional 0.04 25.4 control 4No control 0.04 75.6 5 Fosetyl 0.120% Conventional 0.96 11.0 control 6Potassium 0.112% Conventional 0.06 86.9 phosphate control 7 Conventional1.72 90.0 control 8 No control 0.73 173.0 Note) The concentration of theactive ingredient A is the concentration converted into P₂O₅. Note) Thegrains colored with scarlet or orange inherent for Fusarium head blightand shrink grains were sorted as the diseased grain, and the grainsother than them were sorted as the healthy grains.The concentration of DON contamination of the healthy grains variedalmost within lower range. On the other hand, in the diseased grainswith Fusarium head blight, DON was detected at an extremely highconcentration as expected. However, in the crude wheat determined to bethe diseased grains, difference was observed in the concentration of DONcontamination. That is, in the section given the treatment withpotassium phosphite, the DON contamination concentration was decreaseddepending on the given concentration of potassium phosphite. This way,it is obvious that giving potassium phosphite reduces the DONcontamination concentration in the wheat regardless the presence orabsence or the degree of the disease with Fusarium head blight ofcereals, and further that the reduced degree of the DON contaminationdepends on the given concentration of potassium phosphite.

Example 3 Mixed Use (1)

Wheat (oultivar: Haruyutaka) was seeded on Apr. 23, 2002, and cultivatedaccording to a conventional cultivating standard (Hokkaido, AgricultureDepartment, 1995) to establish test sections with 10 m² per section (3repeats). A suspension containing potassium phosphite and variousfungicidal agents was prepared in a liquid mixture made up of aspreading agent (Gramine S: supplied from Hokkai Sankyo Co., Ltd.) andwater. Using potassium phosphite as the compound A, 0.070% P₂O₅, and the0.006 to 0.025% fungicidal agent for agri-horticulture for the wheat asthe compound B, water solution containing them was prepared, and 100 Lthereof per 10 a was sprayed on leaves in the following growth stages.That is, the first spray (June 24, heading date), the second (June 30,flowering stage), the third (July 9) were performed. The harvest wasperformed on August 12 (50 days after the heading date) by harvestingthe wheat in 3.4 m² in each section. After the harvest, the wheat wasapplied on a rice grader with 2.2 mm opening of. screen to make selectedgrains, which were then pulverized by a rapid pulverizer to make wholegrain powder. The whole grain powder was used as a sample for analysis.The DON contamination concentration was analyzed using HPLC-UV method inofficially fixed methods shown by the Minister of Health, Labor andWelfare. Quantitative analysis was performed by repeating three times,and the quantitative value was an average value thereof.

Preparation of analysis sample solutions and the analysis procedure arebriefly described.

-   -   To 50 g of the whole grain powder, 85% acetonitrile was added,        vigorously agitated for 30 min, and subsequently sonicated for        10 min.    -   After eliminating impurities with filter paper, a filtrate was        purified using a pretreatment column MultiSep #227 to make a        solution for analysis.    -   The test solution for analysis was injected into high        performance liquid chromatography, and DON was detected by        ultraviolet rays.    -   The DON concentration of each test solution was read out from a        standard curve made using DON standard solutions.    -   The incidence rates of Fusarium head blight for Panicles and        spikelets were surveyed for 100 panicles in each section.

The results of the present test are shown in Table 3.

TABLE 3 Mixed use effects of potassium phosphite and various fungicidalagents Presence or Active absence of active ingredient B, DON IncidenceNumber of ingredient A, Concentration concen- rate for diseasedpotassium of active tration panicles spikelets/ phosphite ingredient(ppm) (%) panicle 1 X Tebuconazole 0.39 0.3 0.003 2 ◯ 0.020% 0.38 1.00.010 3 X Metconazole 0.34 0.7 0.007 4 ◯ 0.006% 0.10 1.0 0.010 5 XPropiconazole 1.01 1.0 0.010 6 ◯ 0.017% 0.72 2.7 0.027 7 X Azoxystrobin4.65 2.3 0.027 8 ◯ 0.010% 0.96 2.0 0.023 9 X Kresoxim- 1.05 0.3 0.003 10◯ methyl 0.51 0.7 0.007 0.021% 11 X Iminoctadine 1.04 1.7 0.017 12 ◯acetate 0.20 3.0 0.030 0.025% 13 ◯ 0.77 4.3 0.050 14 Non-treatment 3.696.7 0.138 Note) 0.07% P₂O₅ as potassium phosphite; circle: treated,crisscross: untreated Note) The incidence rates for panicles andspikelet rates were surveyed for 100 panicles in each test section.

Example 4 Mixed Use (2)

Wheat (cultivar: Haruyutaka) was seeded on May 4, 2003, and cultivatedaccording to a conventional cultivating standard (Hokkaido, AgricultureDepartment, 1995) to establish test sections with 10 m² per section (3repeats). A suspension containing potassium phosphite and variousfungicidal agents was prepared in a liquid mixture made up of aspreading agent (Gramine S. supplied from Hokkai Sankyo Co., Ltd.) andwater. Using potassium phosphite as the compound A, 0.070% P₂O₅, and the0.030 to 0.125% fungicidal agent for agri-horticulture for the wheat asthe compound B, an water solution containing them was prepared, and 100L thereof per 10 a was sprayed on leaves in the following growth stages.That is, the first spray (July 1, flowering stage), the second (July 7),the third (July 14) were performed. Harvest was performed on August 25by harvesting all wheat in each test section. After the harvest, using asample divider, the wheat was equally divided, and was applied on a ricegrader with 2.2 mm opening of screen to make selected grains, which werethen pulverized by a rapid pulverizer to make whole grain powder. Thewhole grain powder was used as a sample for analysis. The concentrationof DON contamination was analyzed by the ELISA method in the same way asshown in Example 1.

TABLE 4 Mixed use effects of potassium phosphite and various fungicidalagents Presence or Active absence of active ingredient B, DON IncidenceNumber of ingredient A, Concentration concen- rate for diseasedpotassium of active tration panicles spikelets/ phosphite ingredient(ppm) (%) panicle 1 X Tebuconazole 0.16 7.0 7.3 2 ◯ 0.020% 0.08 5.0 5.03 X Propiconazole 0.57 4.7 5.0 4 ◯ 0.017% 0.11 4.7 5.0 5 X Azoxystrobin0.83 4.3 4.7 6 ◯ 0.010% 0.50 3.7 4.0 7 X Trifloxystrobin 0.35 4.3 5.0 8◯ 0.025% 0.12 3.0 3.0 9 X Iminoctadine 0.22 7.3 8.0 10 ◯ albesilate 0.002.7 2.7 0.030% 11 X sulfur 1.85 8.7 10.3 12 ◯ 0.125% 0.43 8.7 8.7 13 ◯0.57 10.3 11.0 14 Non-treatment 2.15 20.4 23.8 Note) Potassium phosphiteis 0.070% in P₂O₅. ◯: with treatment, X: without treatment Note) Theincidence rates for panicles and spikelets were surveyed for 100panicles in each test section.

In the same way as shown in Example 3, in the mixed use with any of thefungicidal agents for agri-horticulture, both the incidence rate forpanicle and the incidence rate for spikelet exhibited high valuescompared to a single treatment, but the DON contamination amount wasreduced. Worthy of special mention is the result in section given thesingle agent treatment with the compound A, where the disease rate ofFusarium head blight was high, nonetheless, the DON contamination amountwas at a low level.

Example 5 Effects on Fungal Growth and Toxin Production (1)

A water solution containing 5.600% potassium phosphite was prepared,soaked into gas-sterilized wheat seeds of “Hokushin”, and subsequentlyFusarium head blight pathogenic fungus of the cereals, Fusariumgraminearum having DON production capacity was inoculated, which werethen cultured at 27° C. On days 7, 14, 21 and 28 in the culture, theamount of Fusarium head blight pathogenic fungus of the cereals and theamount of DON production present in wheat grains were analyzed.

The preparation of ergosterol analysis sample solutions and the analysisprocedure are briefly described.

-   -   To 5 g of the culture, 80 ml of ethanol was added, and        pulverized using a rapid pulverizer.    -   The ethanol solution containing the pulverized culture was        vigorously agitated for 30 min to extract ergosterol.    -   After eliminating impurities with filter paper, a filtrate was        concentrated under reduced pressure, dried, solidified and        dissolved again in 10 ml of ethanol to make an analysis sample        solution.    -   The analysis sample solution was injected into high performance        liquid chromatography, and ergosterol was detected by        ultraviolet rays.

The preparation of DON analysis sample solutions and the analysisprocedure are briefly described.

-   -   To 4 g of the culture, 80 ml of distilled water was added, and        pulverized using a rapid pulverizer.    -   The water solution containing the pulverized culture was        vigorously agitated for 30 min to extract DON.    -   A part of the extract solution was centrifuged, and the        supernatant was used for the ELISA analysis.    -   According to the instruction manual in the ELISA kit, various        reagents were added, and subsequently an absorbance of each test        solution was measured.    -   The DON concentration of each test solution was read out from        standard curve made using DON standard solution.

TABLE 5 Change over time of bacterial growth and DON production in wheattreated with potassium phosphite Analysis Days of culturing Treatmentitem 7 days 14 days 21 days 28 days Potassium DON (ppm) ND. ND. ND. ND.phosphite Ergosterol 17952 115921 372377 350972 5.6% peak area valueNon- DON (ppm) 6.3 4.43 4.29 37.8 treatment Ergosterol 150453 271882367236 559014 peak area value Note) ND means that the analytical valueis less than the detection limit (0.222 ppm) of the ELISA analysis kit.The amount of ergosterol which was an indicator of the amount of fungiwas continuously increased during the culture in the non-treatmentwhereas reached a plateau on the 21st day in the treatment withpotassium phosphite. With respect to the amount of DON production, inthe non-treatment, the DON production was initiated at an early phase ofthe culture and remarkably increased on the 28th day. On the contrary,in the treatment with potassium phosphite, DON was not detected duringthe entire period of the culture. It is obvious that application ofpotassium phosphite has a high inhibitory effect on the DON productionregardless of the presence or absence or the proliferation degree of thefungi for the Fusarium head blight of cereals.

Example 6 Effects on Fungal Growth and Toxin Production (2)

A water solution containing 0.056% to 2.800% potassium phosphite wasprepared, soaked into gas-sterilized wheat seeds of “Hokushin”, andsubsequently Fusarium head blight pathogenic fungus of the cereals,Fusarium graminearum having DON production capacity was inoculated tothe wheat seeds, which were then cultured at 27° C. On day 28 in theculture, the amount of Fusarium head blight pathogenic fungus of thecereals and the amount of DON production present in wheat grains wereanalyzed by the methods in the same way as shown in Example 5.

TABLE 6 Effects of potassium phosphite concentration on bacterial amountof Fusarium head blight pathogenic fungus of the cereals and the amountof DON production Treatment Ergosterol concentration DON (ppm) peak area0.056% 1.60 1834861 0.112% 0.41 1769582  0.28% ND. 1810454 0.560% ND.1921693 2.800% ND. 85891 Non-treatment 46.1 3601748 Note) ND means thatthe analytical value is less than the detection limit (0.222 ppm) of theELISA analysis kit.

Considering the amount of ergosterol as the indicator of the amount offungi, in the treatment with the water solution of 0.056 to 0.560%potassium phosphite, the inhibitory effect on the fungal amount at amoderate degree and similar degree as compared to that in thenon-treatment (water only) was observed, and the remarkable inhibitoryeffect on the fungal amount was observed in the treatment with the watersolution of 2.800% potassium phosphite. Concerning DON amount, in therange of concentrations from 0.056 to 2.800% of potassium phosphite inthe water solutions, it was significantly lower than that in thenon-treatment. It is obvious that the application of potassium phosphitehas a high inhibitory effect on the DON production regardless of thepresence or absence or the proliferation degree of the fungi for theFusarium head blight of cereals.

INDUSTRIAL APPLICABILITY

According to the invention, it has been demonstrated that an excellentinhibitory effect on mycotoxin contamination, particularly DONcontamination can be had regardless of the presence or absence or thedegree of the disease with Fusarium head blight of cereals although theprevention effect on Fusarium head blight pathogenic fungi of cereals islow by spraying one or more compounds A selected from the groupconsisting of ammonium salts, primary to quaternary ammonium salts,alkali metal salts, alkaline earth metal salts and polyvalent metalsalts of phosphorous acid and phosphite ester onto the growing wheat.Also, it has been demonstrated that the contamination amount ofmycotoxin is further reduced by the mixed use of the other fungicidalagent for agri-horticulture compared to the use of the single fungicidalagent alone.

1. A method for reducing deoxynivalenol contamination in wheatcomprising a step of applying a material consisting essentially ofpotassium phosphite and optionally at least one fungicidal activeingredient for agri-horticulture selected from the group consisting oftebuconazole, metconazole, propiconazole, azoxystrobin, kresoxim-methyl,iminoctadine acetate, iminoctadine albesilate, trifloxystrobin andsulfur to a seed or plant of wheat wherein the amount of potassiumphosphite is sufficient for inhibiting deoxynivalenol production fromplant pathogenic fungi in wheat to 1.1 ppm or less, and is 0.56% byweight or less of said material.
 2. The method according to claim 1,wherein the at least one fungicidal active ingredient foragri-horticulture is selected from the group of azoxystrobin,iminoctadine acetate and iminoctadine albesilate.
 3. The methodaccording to claim 1, wherein the step of applying is to a seed ofwheat.
 4. The method according to claim 1, wherein the step of applyingis to a plant of wheat.
 5. The method according to claim 1, wherein theamount of potassium phosphite is 0.112% by weight or less of saidmaterial.